含P6A寡肽血液的HPLC分离分析  

HPLC analysis of ARPAK in plasma

作　　者：徐友宣[1] 王超[1] 彭师奇[1] 赵明[1] 

机构地区：[1]北京大学药学院,北京100083

出　　处：《中国新药杂志》2003年第7期548-553,共6页Chinese Journal of New Drugs

基　　金：国家科技部"973"重大项目 (G19980 5 1111)

摘　　要：目的 :建立可用于寡肽血液浓度测定并可与ESI MS对接的HPLC方法。方法 :以ARPAK溶栓寡肽为模型化合物 ,考察其对色谱系统的稳定性 ,选择适宜的洗脱系统 ,消除寡肽与血浆蛋白的相互作用 ,确定柱温与寡肽峰面积及保留时间的关系 ,寻找血液中寡肽降解酶抑制剂 ,比较不同的填料对从血液中提取寡肽的影响 ,优化寡肽柱前衍生化的温度、时间、pH环境和试剂。结果 :含ARPAK的小鼠血浆在 5 0℃、pH 8 5的条件下与丹酰氯反应 70min ,衍生化程度可达峰值。含ARPAK的血浆溶液丹酰氯衍生化后用HClO4处理 ,可以有效地使被测的丹酰氯衍生化的ARPAK从蛋白中游离。pH 9 4的磷酸盐缓冲溶液可以有效地阻滞ARPAK在任意时刻的降解。采用自装的XAD 2柱 ,可以定量从鼠血浆中回收ARPAK。ARPAK在A相为 pH 2 1磷酸盐缓冲液 ,B相为乙腈的流动相中显示最佳的分离度。室温进样 ,2 0 %A :80 %B至 4 5 %A :5 5 %B ,10min ;4 5 %A :5 5 %B至 70 %A :30 %B ,1min ,丹酰氯衍生化ARPAK的保留时间为 11 77min ,检测限为 10ng。结论 :上述HPLC条件以及样品前处理方法可用于寡肽血样HPLC/ESI-MS研究。Objection:To establish a HPLC method to be matched for ESI-MS,for determination of oligopeptides in plsama.Methods:ARPAK was adopted as the model peptide,to examine oligopeptide stability to HPLC system,search for suitable elution solvents,to eliminate interreaction of oligopeptide and plasma protein,to determine column temperature and the relationship between peak area and retention time,to search for degradation enzyme inhibitor for oligopeptide in plasma,to compare the effect of column packings on extraction rate of oligopeptide,to optimize the temperature,time,pH value,and reagent needed in pre-column derivatization of oligopeptide.Results:The reaction of ARPAK and dansyl chloride in plasma was completed in 70 minutes at 50℃,and pH 8.5.After the reaction product was treated with HClO 4,the dansy-ARPAK can be quantitatively isolated from the complex formed.Phospate buffer(pH 9.4) solution may inhibit effectively the degradation of ARPAK in plasma.By using XAD-2 column,ARPAK can be separated from plasma.After injection of sample,the retention time of denyl-ARPAK was 11.77 minutes with a detection limit of 10ng.Conclusion:The HPLC conditions and the pre-column treatment procedure may be useful in HPLC/ESI-MS assay of oligopeptide in blood.

关 键 词：ARPAK 荧光衍生化 HPLC分析 

分 类 号：R927.1[医药卫生—药学]